Pelvic organ prolapse (POP) is characterized by weakening of the connective tissues and loss of support for the pelvic organs. Collagen is the predominant, load-bearing protein within pelvic floor connective tissues. In this study, we examined the nanoscopic structures and biomechanics of native collagen fibrils in surgical, vaginal wall connective tissues from healthy women and POP patients. Compared to controls, collagen fibrils in POP samples were bulkier, more uneven in width and stiffer with aberrant D-period. Additionally, the ratio of collagen I (COLI) and collagen III (COLIII) is doubled in POP with a concomitant reduction of the amount of total collagen. Thus, POP is characterized by abnormal biochemical composition and biophysical characteristics of collagen fibrils that form a loose and fragile fiber network accountable for the weak load-bearing capability. The study identifies nanoscale alterations in collagen as diagnostic markers that could enable pre-symptomatic or early diagnosis of POP.